Heart failure (HF) is a major cause of death in the US. A central aspect is reduced cardiac contractility, and much evidence indicates that altered myocyte Ca handling, particularly reduced SR Ca content is centrally responsible. Myocyte Ca & Na regulation are tightly linked by Na/Ca exchange (which we have studied in depth). Our overall goal is to understand altered Ca & Na regulation in HF. We focus on in-depth analysis of key aspects of SR Ca handling (Aim 1 & 2) and myocyte Na transport (Aim 3 & 4), including basic mechanistic & quantitative issues in normal myocytes, and also how these change in HF. We will use our well characterized nonischemic rabbit HF model (& human myocytes) with key mechanistic studies using transgenic and knockout mice. Using interdigitated fluorescence, confocal, electrophysiological and biochemical approaches, we will address 4 issues: 1. Intra-SR free [Ca] ([Ca]sR). Using our new method to directly measure (Ca)sR,we will test a) if important spatial (Ca)sR gradients exist, b) why SR Ca load is low in HF (low (Ca)sR, SR volume or Ca buffering), c) if phospholamban (PLB) reduces SR Ca-ATPase efficiency, and d) how (Ca)sa may dynamically function in terminating SR Ca release during E-C coupling. 2. SR Ca leak in HF and PKA & CaMKII effects. Diastolic SR Ca leak in HF and protein kinase effects are controversial. We will use Ca sparks & our novel method to measure leak. We will clarify how leak is altered in HF as a function of SR Ca load, and how PKA and CaMKII modulate SR Ca leak in control & HF myocytes (including changes in expression & phosphorylation state of SR proteins in HF. 3. Na influx in HF. (Na)+ is elevated in HF and we showed that elevated TTX-sensitive resting Na influx is largely responsible (e.g. vs. Na/H or Na/Ca exchange). We will test whether this is also true during stimulation in HF, and whether the TTX-sensitive Na influx in HF is attributable to slowly inactivating or window Na current. 4. Phospholemman (PLM) and Na/K-ATPase modulation. PLM is an endogenous regulator of Na/K-ATPase, and a major PKA target in heart. We will test hypotheses that a) endogenous PLM inhibits Na/K-ATPase, and that inhibition is relieved by PKA-dependent phosphorylation, b) PLM expression level modulate Na/K-ATPase expression, and c) in HF, lower PLM expression and/or higher phosphorylation explain why lower Na/K-ATPase expression in HF does not depress Na-pump function. These studies will interweave both quantitative fundamental mechanistic studies of cardiac myocyte E-C coupling, Ca and Na regulation in control and HF (regarding SR Ca transport, how SRCa release shuts off during E-C coupling, PKA & CaMKII effects, Na influx, NCX and Na/K-ATPase). We will test explicit mechanistic hypotheses which will enrich our fundamental understanding of Ca and Na regulation in heart cells, but also provide new insight into how these are altered in HF. This should help in developing new therapeutic targets and strategies for the treatment of human HF.